Abstract
Arterial ATP-sensitive K+ (K(ATP)) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K(ATP) channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K(ATP) channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K(ATP) pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K(ATP) channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-beta-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K(ATP) current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K(ATP) channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K(ATP) channel activity.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters / physiology*
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Adenosine Triphosphate / pharmacology
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Adenylyl Cyclases / physiology*
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Animals
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Aorta / enzymology
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Aorta / physiology
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Aorta / ultrastructure*
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Calcitonin Gene-Related Peptide / pharmacology
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Caveolae / chemistry
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Caveolae / drug effects
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Caveolae / enzymology
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Caveolae / physiology*
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Caveolin 1
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Caveolins / analysis
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Caveolins / physiology*
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Cell Compartmentation
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Cell Fractionation
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Cholesterol / analysis
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Cyclic AMP-Dependent Protein Kinases / physiology*
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Glyburide / pharmacology
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Guanosine Diphosphate / analogs & derivatives*
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Guanosine Diphosphate / pharmacology
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Ion Transport / drug effects
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Isoenzymes / physiology
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KATP Channels
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Male
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Membrane Lipids / analysis
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Mesenteric Arteries / chemistry
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Mesenteric Arteries / enzymology
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Muscle, Smooth, Vascular / cytology
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Muscle, Smooth, Vascular / physiology
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Myocytes, Smooth Muscle / chemistry
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Myocytes, Smooth Muscle / enzymology
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Myocytes, Smooth Muscle / physiology*
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Patch-Clamp Techniques
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Peptide Fragments / pharmacology
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Pinacidil / pharmacology
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Potassium / metabolism
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Potassium Channels, Inwardly Rectifying / drug effects
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Potassium Channels, Inwardly Rectifying / isolation & purification
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Potassium Channels, Inwardly Rectifying / physiology*
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Propranolol / pharmacology
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Rats
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Rats, Wistar
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Sphingolipids / analysis
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Theophylline / analogs & derivatives*
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Theophylline / pharmacology
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Thionucleotides / pharmacology
Substances
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ATP-Binding Cassette Transporters
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Cav1 protein, rat
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Caveolin 1
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Caveolins
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Isoenzymes
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KATP Channels
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Membrane Lipids
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Peptide Fragments
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Potassium Channels, Inwardly Rectifying
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Sphingolipids
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Thionucleotides
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uK-ATP-1 potassium channel
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calcitonin gene-related peptide (8-37)
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Guanosine Diphosphate
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guanosine 5'-O-(2-thiodiphosphate)
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Pinacidil
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8-(4-sulfophenyl)theophylline
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Adenosine Triphosphate
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Cholesterol
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Propranolol
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Theophylline
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Cyclic AMP-Dependent Protein Kinases
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Adenylyl Cyclases
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Calcitonin Gene-Related Peptide
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Potassium
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Glyburide